The long term goals of this proposal are to understand the mechanism and regulation of bovine papillomavirus (BPV) DNA replication. Study of this virus is important for two reasons.First, the BPV system can provide information of the mechanism and regulation of human chromosomal DNA replication. Second, the closely related human papillomavirus is the causal agent of very common sexually transmitted diseases, and this virus has been strongly implicated as a cause of certain human cancers. Thus, study of these viruses will allow the development of reagents that prevent the replication and the subsequent propagation of these viruses in humans.The specific aims of this project are five-fold. First, the essential domains within the BPV origin of replication will be characterized.The BPV ori will be subjected to site-directed mutagenesis and the mutant ori DNA molecules then tested for activity using three assays: the ori-unwinding reaction dependent on the BPV El protein, DNA replication in vitro, and DNA replication in vivo. Second, the molecular interactions between ori and the BPV E1 and E2 proteins will be determined by enzymatic and chemical probing of the protein-DNA complexes.Third, the multimeric structure of E1 bound to ori will be characterized using scanning transmission electron microscopy. The effect of the BPV E2 protein and ATP on the oligomeric state of E1 bound to ori will be examined. Fourth the two- hybrid system will be used to identify proteins from a mouse cDNA library that can interact with the E1 protein in vivo. The sequences of the cDNA molecules will be determined. Fifth, the cell-cycle regulation of the interaction of the BPV E1 and E2 proteins with ori will be investigated. The amount of protein binding to ori and the presence of structural changes within ori will be probed chemically and enzymatically in non-synchronized cells and in elutriated cells. The effect of extracts of elutriated mouse cells on the binding of the E1 and E2 proteins to ori and on the induction of ori structural changes will also be examined.